1. Field of the Invention
This invention generally relates to the field of HDL-associated proteins and paraoxonase activity. More specifically, the invention provides a method of modulating paraoxonase activity using apolipoprotein A-I mutant peptides having cysteine substitutions and polynucleotides encoding same. The invention has further application in the fields of treating and preventing cardiovascular diseases.
2. Description of the Related Art
Elevated HDL concentration has been correlated with a reduced risk for coronary artery disease (CAD). HDL particles are thought to possess the ability to attenuate the initiation and progression of atherosclerotic lesions. It has been proposed that the protective effects of HDL are related, in part, to its ability to inhibit the formation of minimally oxidized LDL, thus preventing the accumulation of proinflammatory mediators associated with the onset of atherogenesis. This antioxidative activity is ascribed to the enzyme, Paraoxonase (PON), which is exclusively transported on plasma HDL. HDL-associated PON possesses peroxidase-like activity that can likely protect against lipoprotein oxidation.
Human paraoxonase (PON or PON1A) is a 43 kDa glycoprotein with a broad specificity class A esterase activity (La Du, B. N., et al., (1993) Chem Biol Interact 87, 25–34), capable of hydrolyzing a broad spectrum of organophosphate substrates and a number of aromatic carboxylic acid esters (Gan, K. N., Smolen, A., Eckerson, H. W., and La Du, B. N. (1991) Drug Metab Dispos 19, 100–6.).
Recent studies suggest that this enzyme's arylesterase activity can hydrolyze bioactive oxidized phospholipids (Watson, A. D., et al., (1995) J Clin Invest 96, 2882–91). In vitro studies by Mackness et al. (Atherosclerosis (1993) 104, 129–35) have demonstrated that HDL with PON is capable of attenuating the production of lipid hydroperoxides on LDL and the formation of minimally oxidized LDL.
In PON knockout mice there is an accumulation of lipid hydroperoxides on LDL, and these mice are more susceptible to diet-induced atherogenesis (Shih, D. M., et al., Nature 394, 284–7, 1998). Furthermore, in humans, low concentrations of PON in plasma have been correlated with an increased risk for CAD (McElveen, J., et al., (1986) Clin Chem 32, 671–3. 11; Navab, M., et al., (1997) J Clin Invest 99, 2005–19). Such studies suggest an important role for HDL-PON in the protection of LDL from oxidation and the concomitant protection of the artery wall from atherogenesis. It is likely that increased transport and/or stability of the PON enzyme on HDL increases HDL's protective properties.
Apolipoprotein A-I is the major structural protein on HDL. It consists of a series of amphipathic helices that are functionally important for protein-lipid interactions as well as protein-protein interactions. The carboxy terminus of ApoA-I has high lipid-binding capacity, while the amino terminus has limited lipid-binding capacity but may be important in protein-protein interaction (Frank, P. G., and Marcel, Y. L. (2000) J. Lipid Res. 41, 853–72.). ApoA-I is largely responsible for mediating HDL assembly and is a determinant of HDL structure and composition.
The presence of a cysteine mutations in Apolipoprotein A-I protein at positions 151 and 173 are known in the art for their anti-atherogenic chracteristics. The ApoA-IMilano mutation (R173C) was first described by Franceschini G., et al., J Clin Invest 1980; 66:892–900. Carriers of the ApoA-IMilano trait characterized by the presence of a sulfhydryl group in ApoA-I (Arg173→Cys mutation) have substantial reductions in ApoA-I (approximately 50%) but have no predisposition for CAD (Franceschini, G., et al., (1980) J Clin Invest 66, 892–900). These patients have PON mass and activity similar to normal subjects. Sirtori et al., in U.S. Pat. No. 5,876,968, describe a pharmacological composition comprising the disulfide bonded dimer of Apolipoprotein AIMilano in a substantially pure form, and methods of making and purifying the dimer. Abrahamsén et al., in U.S. Pat. No. 5,721,114, describe an expression system and methods of producing Apolipoprotein A-IMilano.
Another Arg→Cys variant, Arg151→Cys (ApoA-IParis) (Bruckert, E., et al., (1997) Atherosclerosis 128, 121–8), has low HDL cholesterol and ApoA-I without increased CAD. Benoit et al., relate variants of apolipoprotein A-I comprising a cysteine at position 151 (ApoA-IParis) or 175 and means for expressing these variants, in U.S. Pat No. 6,258,596.
There may be direct interactions between PON and ApoA-I. Recent studies by Sorenson et al. (Arterioscler Thromb Vasc Biol (1999) 19, 2214–25), using recombinant WT PON and a PON mutant lacking the hydrophobic Apolipoprotein A-I and Paraoxonase Activity N-terminal domain, suggested that PON interacts with HDL primarily through interactions of this hydrophobic moiety with HDL phospholipid. ApoA-I did enhance enzyme stability, again suggesting that on the HDL particles there may be PON-ApoA-I interactions.
Despite the strong association between HDL and PON, deficiencies in HDL and ApoA-I show variable PON activity and mass. Homozygous Tangiers patients and ApoA-IPisa patients have ApoA-I concentrations <5% of normal, and some of these individuals have documented coronary artery disease (Miccoli, R., Circulation (1996) 94, 1622–8). This condition is associated with a 60–75% decrease in PON mass and PON arylesterase activity (James, R. W., (1998) Atherosclerosis 139, 77–82); potentially the reduction in PON may be a contributing factor to the coronary heart disease noted in the patients. Patients heterozygous for the Lys107→0 mutation (ApoAIHelsinki) (Tilly-Kiesi, M., (1995) Arterioscler Thromb Vasc Biol 15, 1294–306) have a 30% reduction in ApoA-I and approximately 40% reduction in PON plasma concentration (James, R. W., et al., (1998) Atherosclerosis 139, 77–82), and some subjects have documented coronary disease.
Like ApoA-IMilano and ApoA-IParis, PON has a free sulfhydryl group, and site-directed mutagenesis has ruled out the possibility that this sulfhydryl group is part of the enzyme's esterase catalytic site (Sorenson, R. C., et al., (1995) Proc Natl Acad Sci USA 92, 7187–91). Potentially the presence of a Cys in ApoA-IMilano (and by extension ApoA-IParis) could alter HDL, leading to increased PON stability or activity.